In a number of applications such as gene analysis and DNA profiling, it is desirable to multiply the amount of particular nucleic acid sequences present in the sample. For example, a duplex DNA segment of up to 5,000 base pairs in length may be amplified many million-fold by means of the polymerase chain reaction (PCR;[1]), starting from as little as a single copy [2,3].
In this technique a denatured duplex DNA sample is incubated with a molar excess of two oligonucleotide primers, one being complementary to a short sequence in one strand of the DNA duplex and the other being identical to a second short sequence upstream of it (i.e. more 5'), such that each primer anneals to its complementary sequence and primes the template-dependent synthesis by a DNA polymerase of a complementary strand which extends beyond the site of annealing of the other primer, through the incorporation of deoxynucleotide triphosphates provided. Multiple cycles of denaturation, annealing and synthesis each afford an approximate doubling of the amount of target sequence, where the target sequence is defined as the DNA sequence subtended by and including the primers. Each cycle is controlled by varying the temperature to permit successive denaturation of complementary strands of duplex DNA, annealing of the primers to their complementary sequences and primed synthesis of new complementary DNA strands. The use of a thermostable DNA polymerase obviates the necessity of adding new enzyme for each cycle [4,5], thus allowing automation of the DNA amplification process simply by thermal cycling. Twenty amplification cycles increase the amount of target sequence by approximately one million-fold (being theoretically 2.sup.20 but usually less in practice).
The oligonucleotide primers used to prime DNA synthesis in the polymerase chain reaction need not necessarily complement sequences within the DNA of the sample but may be complementary to oligonucleotides that have been ligated to the termini of DNA fragments generated from the sample DNA by digestion with a restriction endonuclease or other means [6].
The polymerase chain reaction can be implemented with thermostable DNA polymerases isolated from a number of different sources and with enzymatically-active fragments and derivatives of naturally occurring thermostable DNA polymerases. In its most usual application the polymerase chain reaction is used to amplify DNA target sequences but it can be used also to provide amplified DNA sequences corresponding to RNA target sequences by first synthesizing DNA sequences complementary to the target RNA sequences. This is achieved by primed synthesis with a class of DNA polymerase enzyme known as reverse transcriptase. Reverse transcriptases are also capable of synthesizing DNA complementary to DNA templates and so may be used for primed DNA synthesis in the polymerase chain reaction. In particular a thermostable reverse transcriptase may be used for this purpose.
For the purposes of DNA analysis the polymerase chain reaction technique offers the advantage of providing a large amount of a specific sequence of DNA, whose extremities are defined by the included primers, sufficient for detailed analysis. More detailed information regarding the polymerase chain reaction technique can be found in Innes et al 1990 [7].
A further thermal cycling technique that is used to amplify specific target nucleic acid sequences is the ligation amplification reaction (LAR). For exponential amplification with this technique, two pairs of oligonucleotides that are complementary to overlapping continuous portions of the complementary strands of a target sequence in sample DNA are ligated together in a template-dependent reaction catalysed by a DNA ligase enzyme. The duplex strands of DNA are then denatured by heating and the specific oligonucleotides again are allowed to anneal and are again ligated. Multiple cycles of denaturation, annealing and ligation each afford an approximate doubling of the amount of target sequence [8]. This procedure has particular application in discriminating between alleles that may differ by as little as a single base in their respective DNA sequences and is particularly useful in analysing the sequence of DNA products resulting from amplification of target sequences by the polymerase chain reaction [8]. The use of a thermostable DNA ligase in the ligation amplification reaction allows the procedure to be automated by thermal cycling.
Devices for use in the automated thermal cycling of reaction mixtures for amplification of nucleic acid sequences typically consist of a heat conductive material provided with vertical cylindrical channels to receive vessels in which the reaction is to take place. The vessels typically are small plastic tubes with a conical lower section and an attached lid, typically capable of holding 0.5 ml or 1.5 ml of liquid and known as "Eppendorf tubes". The heat conductive material is provided with heating/cooling means. One of the difficulties encountered in the use of such devices has been the achievement of rapid cooling of the reaction mixture. The most common solutions to this problem have been to use circulation of cold water, a standard refrigeration device, or a Peltier effect heat pump. The last named is the most desirable option but it suffers from the drawback that continual cycling between heating and cooling typically leads to failure of the Peltier effect heat pump.
Many present applications of nucleic acid amplification procedures in general and of the polymerase chain reaction in particular (and the anticipated area of most intensive application) is its use in routine, repetitive analysis of a particular sequence of genomic DNA that may be a gene or may be linked closely to a gene having sequence variants of known deleterious or beneficial effect ("gene diagnosis"). Such repetitive analyses of a single target sequence and its variants are particularly suited to automation.
Nucleic acid amplification procedures are not restricted to qualitative identification of specific nucleic acid sequences but can also be used to quantify the amount of particular nucleic acid sequences present in a sample. In particular they are commonly used to quantify the amount of RNA sequences present in a sample [9,10]. The application of thermal cycling amplification procedures to accurate quantitative analysis of nucleic acid sequences requires that all samples be exposed to identical thermal cycles, a requirement that is not met with presently available means for thermal cycling of sample reaction mixtures.
The most critical problem in applying nucleic acid amplification procedures, particularly in repetitive diagnostic assays in a clinical laboratory, is contamination of the reaction mixture by small amounts of DNA which contain sequences capable of being amplified under the conditions of assay. The products of previous amplification reactions present a particular problem by virtue of their abundance and the fact that they comprise specific target sequences. Automation of procedures minimise the opportunity for such contamination and allows the implementation of stringent quality control.